University of Groningen Synthesis and characterization of ...Chapter 1 9 Human gut microbiota The...
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University of Groningen
Synthesis and characterization of lactose and lactulose derived oligosaccharides byglucansucrase and trans-sialidase enzymesPham, Thi Thu Hien
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7
Chapter 1
Introduction
Chapter 1
8
Human gastrointestinal tract
The human digestive system is a complex series of organs and glands that processes
food (Figure 1). After entering the mouth with physical breakdown by chewing,
food continues its way through stomach and intestine where it is partly digested by
human digestive enzymes, i.e. salivary enzymes, pancreatic enzymes and enzymes
excreted in the small intestine. The undigested food ends up in the large intestine or
colon where it is fermented by various microorganisms. The gut microbiome is the
largest microbial community of the human body with approximately 1,000 bacterial
species; most of the gut microbiome resides in the large intestine.1 A healthy gut
microbiome provides a barrier against colonization by pathogens through
competition, assists the GI tract by degradation of complex nutrients providing
energy and essential vitamins, contributes to lipid metabolism and lipid absorption
by lowered pH as a result of short-chain fatty acids secretion, and stimulates the
immune system.2,3
Figure 1: Scheme of the human digestive system (source: www.Storyblocks.com).
Chapter 1
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Human gut microbiota
The composition of the intestinal microbiota of infants is largely regulated by the
diet.4 At birth the digestive tract of human is sterile and soon after becomes
colonized by microbes originating from the mother's vagina and feces, as well as
from the environment. The infant may be fed by breast milk or an alternative source
like formula milk, resulting in different microbiota compositions. With breast-fed
infants, gut microbiota composition is more dominated by bifidobacteria; in contrast,
formula-feeding without added health beneficial oligosaccharides leads to the
development of a gut microbiota with a more adult type of distribution.5,6,7 Breast-
fed infants show significantly higher counts
of Bifidobacterium and Lactobacillus and lower counts of Enterobacteriaceae,
Clostridium coccoides group, Staphylococcus and Bacteroides compared with
formula-fed infants.8,9,10 Bifidobacteria and lactobacilli are considered the most
important health-beneficial bacteria for the human host, whereas staphylococci and
clostridia are potentially pathogenic.11
Human breast milk thus is an important source of oligosaccharides for the neonate’s
developing microbiota.12 The intestinal microbiota is known to be very important for
the development of the gut physiology and the immune system. Attempts have been
made to mimic the intestinal microbiota of breast-fed infants by formula-feeding.
The composition of the intestinal microbiota can be influenced either by
administration of health-promoting bacteria, so-called probiotics, or the dietary
ingredients, so called prebiotics.13 They have shown beneficial effects in infants’
health, providing protection against infections,14,15,16 that can cause diarrhea,17,18 and
necrotizing enterocolitis,19 as well as reducing atopic dermatitis.20,21,22 Probiotic
bacteria, which are able to survive the gastrointestinal tract exert their biological
activity by interaction with the surface of the small intestine, and colonize the colon.
The most common probiotic bacteria that have been studied and used are
Chapter 1
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bifidobacteria or lactobacilli. However, to maintain colonization it is essential to
keep them alive.23 Upon ingestion they are confronted with physical and chemical
barriers such as gastric acid, bile acids. Reaching the colon, they still have to
compete for nutrients and colonization sites with the host’s resident species. As a
result, a small proportion of ingested probiotic bacteria successfully colonizes the
colon.24,25,26 An alternative approach which partly overcomes the limitations of
probiotics is the use of prebiotics.27 Prebiotics are generally defined as “a substrate
that is selectively utilized by host microorganisms conferring a health benefit”.28
Those substrates that are non-digestible during the passage through the small
intestine without being absorbed or utilized, reach the colon, and stimulate
selectively health promoting colonic bacteria.29
Human milk oligosaccharides
The best known natural prebiotic compounds are oligosaccharides from human
breast milk. Human milk oligosaccharides (hMOS) have been well studied and
documented for their prebiotic, and particularly bifidogenic effects.30,31,32,33,34 In
human milk, free oligosaccharides comprise the third most abundant component
after lactose and fat, reaching levels of approximately 5 - 20 g L-1.35,36 The
concentration of hMOS is not constant over time, and tends to decrease during
lactation.37 Human milk oligosaccharides are built up from five monosaccharide
building blocks; i.e. glucose (Glc), galactose (Gal), N-acetylglucosamine (GlcNAc),
Fucose (Fuc), and N-acetylneuraminic acid (Neu5Ac). The structural composition of
hMOS always starts with a lactose core at the reducing end. Lactose can be
elongated with lacto-N-biose units (β-Gal-(1→3)-GlcNAc; Type 1) or lactosamine
units (β-Gal-(1→4)-GlcNAc; Type 2) (Figure 2). The Type 1 core structure of
hMOS can be further elongated in linear or branched form. Lactose and elongated
lactose of hMOS can be further functionalized with fucose and/or sialic acid.38
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Figure 2: Schematic structures of hMOS.
An alternative source for hMOS in nature is currently not available. Milk of
domesticated dairy animals does not match the large amount and high structural
diversity of hMOS.39 Content of milk oligosaccharides in human milk is 100 to 1000
fold higher than MOS in milk of most domesticated animals including cows, goats,
sheep and pigs.40,41,42 Although these milks have a higher relative abundance of
sialylated oligosaccharides (up to 90% of all MOS),43 there are more acidic
oligosaccharides in human milk in terms of mg mL-1.43 In bovine milk, which has
been used as basis for infant formula milk, approximately 70 % of MOS are
sialylated compared to 10 - 20 % in hMOS. But there are only approximately 0.03-
0.06 g L-1 free oligosaccharides in bovine milk compared to 5 - 20 g L-1 in human
milk, which amounts to 0.5 - 4 g L-1 sialylated hMOS.44,45 Moreover, domesticated
animal MOS contain not only Neu5Ac but also some of the non-human sialic acid
derivative N-glycolylneuraminic acid (Neu5Gc). Nowadays, many babies have
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limited access to human milk and receive infant formula as a replacement. Currently
used bovine milk based infant formula lacks the abundance and complexity of
oligosaccharides that human milk provides, and is enriched with synthetic
prebiotics, which do not possess yet the advanced functionality of hMOS.38
Synthesis of real hMOS or structurally/functionally effective hMOS mimics thus is
highly interesting for application in infant formula.
Prebiotics
Non-digestible carbohydrates (NDCs) have received a lot of attention as candidates
to apply in infant formula to mimic molecular size and prebiotic functions of
hMOS.46,47,48,49,50 They are complex carbohydrates with a molecular size mostly
ranging from 3 to 10 sugar moieties. There are several cases of very high DP up to
60 like inulin or very low down to 2 like lactulose.51 Their structural compositions
contain sugars in α- or β-configuration, linear or branched chains that may play an
essential role for their indigestibility in the upper parts of the intestine of the host.52
A number of saccharides has been explored for their prebiotic potential, the most
well-known prebiotic is the mixture of 90% Galacto-oligosaccharides (GOS) and
10% fructo-oligosaccharides (FOS) which has been selected for use in infant
formula milk to mimic the prebiotic effects of neutral human milk
oligosaccharides.46,53 GOS comprise a mixture of galactosyl moieties linked with
(β1→2), (β1→3), (β1→4), or (β1→6), with various sizes (mostly DP2 - DP5)
(Figure 3).54,55 The composition of the GOS mixture highly depends on the source of
the β-galactosidase used for their synthesis using lactose as acceptor substrate.54,56
There are currently several GOS products on the market such as Vivinal® GOS,
Oligomate 55 and Bimuno.57,58,59 This group of oligosaccharides has been widely
studied and shown to have stimulatory effects on the growth of probiotic bacteria to
various extents.60,61,22 Another group of oligosaccharides that has attracted much
commercial interest as prebiotics are FOS. These oligosaccharides can be obtained
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from natural sources like chicory root derived inulin or synthesized enzymatically
from sucrose by bacterial fructansucrase enzymes.62,77 Inulin normally consists of a
sucrose core with one or more (β2→1) linked fructosyl unit elongations, but there is
another type of FOS lacking the terminal glucose part of the sucrose. The degree of
polymerization of FOS varies between 2 and 60 units (Figure 3).63 The stimulatory
effect toward bifidobacteria (Bifidogenic effects) of FOS have been widely
studied.64,65
Figure 3: Schematic structures of GOS, FOS and Lactulose as examples of prebiotic
compounds.
The most common disaccharide used as prebiotic is lactulose. This is a synthetic
disaccharide in the form Gal(β1→4)-Fru (Figure 3). Lactulose was shown to be
resistant to digestion in the small intestine, and showed selective stimulation towards
growth of lactobacilli and bifidobacteria.66,67,68,69 These are prebiotics with well-
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known status supported by a significant number of human, double-blind and placebo
controlled trials.70,71 However, there has been a growing search for new
carbohydrates which could be considered as emerging prebiotics such as
lactosucrose; isomalto-oligosaccharides; resistant starch; xylo-oligosaccharides,
arabinoxylo-oligosaccharides and pectic-oligosaccharides.71,72,73,74,75,76 Where
studied, most of these prebiotics however lack the pathogen exclusion and immune-
and barrier modulating effects that hMOS possess.
Synthesis of Prebiotics and hMOS mimics
Prebiotics and hMOS mimics can be either chemically or enzymatically synthesized.
However, chemical synthesis is cumbersome because it requires many synthetic
steps and a lot of effort to get rid of side products.77 The high selectivity and regio-
specificity of enzymatic routes has advantages over the chemical approach.77,78 The
microbial whole cell engineered biosynthetic routes, with outstanding features to
scale-up for economic production, appeared to be the preferred choices to produce
hMOS compounds like 2'-fucosyllactose (2'-FL).79,80 However, prebiotic synthesis
using whole cell biosynthetic approaches requires a rigorous removal of the
production strain before their application in infant food, and a clear proof that no
genetically modified organisms remain is challenging. Application of isolated and
highly specific enzymes for synthesis of oligosaccharides may simply overcome this
obstacle. In addition, it is easier to control various incubation conditions, such as
reaction conditions (enzyme/substrate concentrations) and environmental conditions
(pH, temperature, metal ion), when using enzymes for synthesis of hMOS mimics
compared to the whole-cell biocatalysts.81 From a practical viewpoint, glycosidase
enzymes are the preferred choice, they are generally more available, and less
expensive than glycosyl-transferases, and do not require expensive nucleotide-sugar
donors.82 The choice of suitable substrates and highly active glycosidases clearly
plays a key role in allowing the synthesis of ‘tailor-made’ hMOS mimics of high
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interest for application in the food industry. Lactose is always at the reducing end of
human milk oligosaccharides, this compound is considered as the initial substrate for
hMOS synthesis.83 Moreover, galactose is present in a high content in hMOS. Thus,
lactose and lactose derivatives like GOS are potential candidates for trans-
glycosylation to mimic hMOS.
Glucansucrase and trans-glycosylation
Glucansucrases belong to glycoside hydrolase family 70 (GH70)
(http://www.CAZy.org) and are extracellular trans-glycosidases found in lactic acid
bacteria.84,85 GH70 glucansucrases belong to the α-amylase superfamily based on
amino acid sequence similarity and structure analogy.86 They are structurally and
mechanistically related to GH13 and GH77 enzymes.87 To date, three-dimensional
structures of four microbial glucansucansucrases were obtained by crystallization of
the recombinantly produced and truncated forms of these proteins, including those
from Lactobacillus reuteri 180,88 L. reuteri 121,89 Streptococcus mutans,90 and
Leuconostoc mesenteroides NRRL B-1299.91
The three-dimensional structures of truncated glucansucrases revealed that they
exhibit a U-type shape and are organized into five domains (A, B, C, IV and V). All
the domains except domain C are made up from discontinuous segments of the
polypeptide. The catalytic domain adopts a (β/α)8 barrel fold and harbors a catalytic
triad, which is composed of two aspartates and one glutamate.87 An N-terminal
domain of variable length and a C-terminal putative glucan-binding domain flank
the central catalytic domain in these glucansucrase enzymes.85
GH70 enzymes follow a double-displacement reaction mechanism, and possess 3
catalytic residues, D1025 (nucleophile), E1063 (acid/base) and D1136 (transition
state stabilizing residue) (Gtf180-ΔN numbering). The reaction starts with cleavage
of the (α1→2) bond of sucrose yielding a covalent glucosyl-enzyme intermediate.
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This is followed by binding of the acceptor substrate and transfer of the covalently
bound glucosyl residue to the acceptor molecule, forming a new glycosidic
linkage.88 The anomeric configuration of the donor is conserved in the product.92,93
Glucansucrase enzymes in family GH70 transfer glucose from sucrose to the non-
reducing end of oligosaccharides in a processive manner, retaining the α-
regiospecificity.94 Depending on the nature of the acceptor substrate, glucansucrase
enzymes catalyze three types of reactions: hydrolysis of sucrose with water as
acceptor, polymerization with growing α-glucan chains as acceptor, or trans-
glycosylation with sucrose as donor substrate and other compounds as acceptor
substrates (including oligosaccharides).87 The glucosidic linkage type formed in the
product is dependent on the acceptor substrate and the enzyme specificity.
Glucansucrases are capable of producing α-glucans with various linkage types,
namely dextran, containing mainly (α1→6) linkages; mutan, consisting
predominantly of (α1→3) linkages; alternan, comprising alternating (α1→6) and
(α1→3) linkages; and reuteran, containing (α1→4) and (α1→6) linkages.85,95 Only
the branching glucansucrase Dsr-E from Leuconostoc mesenteroides NRRLB-1299
can introduce single (α1→2) glucosyl branches in a dextran backbone.96,97 Gtf180-
ΔN produces an α-glucan with 69% (α1→6) and 31% (α1→3) glycosidic linkages
while GtfA-ΔN produces an α-glucan with 58% (α1→4) and 42% (α1→6)
glycosidic linkages.98,99
These enzymes synthesize not only α-glucan polymers but also efficiently catalyze
transfer of glucose moieties from sucrose as donor substrate to numerous hydroxyl-
group containing molecules.100,101,102,103,104,105 In case of these small sugar acceptor
substrates, low molecular mass oligosaccharides are synthesized with different types
of linkage, size, branching, and physicochemical properties.106 Maltose is considered
to be the most effective acceptor substrate of glucansucrase enzymes, synthesizing
various products (DP 3-6) such as panose or other isomaltooligosaccharides.107,108,109
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Other acceptor substrates that were studied include isomaltose, nigerose, methyl α-
D-glucoside, 1,5-anhydro-D-glucitol, D-glucose, turanose, methyl β-D-glucoside,
cellobiose, and L-sorbose.110
Lactose, raffinose, melibiose, D-galactose, and D-xylose are also used as acceptor
substrate by these Gtf enzymes but only give a single glucosylated product each.110
More recently it was reported that dextransucrases from Leuconostoc mesenteroides
and Weissella confusa also use lactose as their acceptor substrate synthesizing 2-α-
D-glucopyranosyl-lactose.111,112 Beside carbohydrates, glucansucrase enzymes are
also able to use non-carbohydrates as their acceptor substrates, i.e. L-ascorbic acid,
luteolin, catechol and various phenolic compounds.82,101,103,104,113 Because of their
diverse product structures in terms of α-glycosidic linkage types, molecular size,
branching and physico-chemical properties, glucansucrases have attracted increasing
interest for industrial applications in food, medicine, cosmetics etc.114
The most common application of α-glucans is the use as sweetening, stabilizing,
viscosifying, emulsifying or water-binding agents in food as well as non-food
industries.115,116,117,118 Moreover, α-glucans and oligosaccharides synthesized by
glucansucrases have shown evidence of prebiotic properties, stimulating growth of
beneficial intestinal bacteria such as Bifidobacterium and Lactobacillus.119 Isomalto-
oligosaccharides (IMOs) are composed of glucose monomers linked by (α1→6)
glucosidic linkages, and have been widely studied as potentially prebiotic.119,73,120
Another group of gluco-oligosaccharides, which are synthesized by glucansucrases
from Leuconostoc mesenteroides using sucrose as donor substrate and maltose as
acceptor substrate, also has potential stimulatory effects on gut bacteria.121,122 In
another study, the addition of an α-glucan product to animal feed improved the
weight gain of piglets and broilers.123 A lactose-derived trisaccharide compound, i.e.
2-glucosyl-lactose, synthesized by L. mesenteroides dextransucrase using lactose as
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acceptor substrate showed selective stimulatory effects on growth of
Bifidobacterium breve.111
Prebiotic effects of gluco-olicosaccharides were shown to be inversely dependent on
the size of the oligosaccharides synthesized by alternansucrase and dextransucrase,
with DP3 possessing the highest prebiotic potential towards bifidobacteria i.e. B.
bifidum, B. longum, B. angulatum.124,125 Therefore, α-glucans and oligosaccharides
synthesized by glucansucrases with a large variety of structures hold great potential
for food applications, more particularly for prebiotic applications.
Trans-sialidase
In human milk, lactose and hMOS backbones can be decorated with sialic acid to
become acidic oligosaccharides.38 There is increasing evidence for the functional
effects of this group of oligosaccharides on human health.126,127,128,129 Sialylated
oligosaccharides are able to prevent intestinal attachment of pathogens by acting as
receptor analogs competing with epithelial ligands for bacterial binding.130,131,132,133
Binding of Cholera toxin was inhibited by 3′-sialyllactose.134 An individual
sialylated hMOS structure, disialyllacto-N-tetraose (DSLNT), contributes to the
protective effects against one of the most common and fatal intestinal disorders in
preterm infants, i.e. necrotizing enterocolitis (NEC).129 Sialylated hMOS have also
been indicated as important factors in brain development, sialic acids increase the
production of gangliosides, which are important components of membrane receptors
and cell surfaces of the nervous system.135 The structure 3'-sialyllactose has been
shown to induce the growth of various common probiotic bacteria including the
infant gut-related Bifidobacterium longum subsp. infantis, B. longum 232, B.
infantis 233, B. infantis 1497 and B. lactis HN019.136 In view of their important
functions, enzymatic synthesis of these acidic oligosaccharides for application in
infant formula has attracted interest.
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The trans-sialidases (EC 3.2.1.18) are glycosidases that naturally catalyze the
transfer of sialyl residues from one sialo-glycan to the terminal Gal residue of
another asialo-glycan.137 In micro-organisms, these enzymes are virulence factors
that enable spreading and infection of host cells.138 Trans-sialidase was first
identified in and isolated from Trypanosoma species. Trans-sialidase from
Trypanosoma cruzi preferentially catalyzes the reversible transfer of (α2→3)-linked
sialic acids from donor glycans directly to terminal β-Gal-containing acceptor
molecules, thereby giving rise to new (α2→3) glycosidic linkages (Figure
4).139,140 When the acceptor substrate is absent, the enzyme acts as a hydrolase
transferring sialic acid to water.137 Trans-sialidase from T. cruzi (TcTS) has been
best documented. The TcTS enzyme has been suggested to be involved in the
mammalian host cell invasion and pathogenesis of T. cruzi leading to Chagas
disease.137,141 In T. cruzi, surface sialylation to scavenge sialic acid plays a crucial
role for their adhesion and invasion to the host cell.142 The recombinant TcTS
enzyme catalyzes the transfer of sialic acid from donor to acceptor with retention of
the configuration of the sialyl glycosidic linkages.143 Trans-sialidase from
Trypanosoma generally has a wide variety of acceptor substrate specificities, albeit
that they favor oligosaccharides and glycoproteins.144,145,146 Recently, casein
glycomacropeptide (GMP), an affordable source of sialic acid, was used in the
synthesis of sialylated galacto-oligosaccharides.147,148 GMP is the soluble
glycosylated casein residue produced by chymosin action on κ-casein during the
cheese manufacturing process. The O-glycans on GMP comprise of Neu5Ac-
containing components including major elements Neu5Ac(α2→3)-Gal(β1→3)-
GalNAc and Neu5Ac(α2→3)-Gal(β1→3)-[Neu5Ac(α2→6)]GalNAc, which can be
used as donor substrates.149,150 Trans-sialidase from T. cruzi is known to be specific
for terminal β-galactosyl residues, any compounds possessing a terminal β-Gal
residue can be used as acceptor substrate.151 However, it has been reported recently
that sialylation of non-terminal β-Gal residues in GOS can also be catalyzed by
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TcTS, provided that two Gal-residues are linked together with a (β1→6)
linkage.136,152
Figure 4: Reversible trans-glycosylation of (α2→3)-linked N-acetylneuraminic acid
between Neu5Ac-(α2→3)Gal-OR1 and Neu5Ac-(α2→3)Gal-OR2, catalyzed by
trypanosomal trans-sialidases.137
Outline of the thesis
Health beneficial oligosaccharides are of great interest for industry and society.
Synthesis of prebiotic oligosaccharides are explored using a wide variety of
methods. Enzymatic synthesis using cheap and available substrates and enzymes
provides clear benefits for scale-up of the production. Glucansucrases are known as
efficient catalysts for synthesis of α-glucans and other gluco-oligosaccharides.
Relatively little is known about their ability to use lactose and galacto-
oligosaccharides (GOS) as acceptor substrates. The aim of this PhD project was to
provide more insights into the activity and product specificity of glucansucrases
Gtf180-ΔN and GtfA-ΔN when acting on these acceptors with focus on product
structural analysis and their possible selective stimulatory effects on growth of gut
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bacteria. Chapter 1 reviews the current literature and knowledge about health
beneficial oligosaccharides including hMOS and the enzyme biocatalysts used,
glucansucrase of Lactobacillus reuteri and trans-sialidase from Trypanosoma cruzi.
In chapter 2, we investigated the ability of the Gtf180-ΔN and GtfA-ΔN enzymes to
use lactose as acceptor substrate for trans-glucosylation, using sucrose as donor
substrate. The results showed that both enzymes synthesized similar transfer
products with a degree of polymerization (DP) of 3 to 4, therefore called GL34
mixture. New linkage types were observed when using lactose as acceptor than
observed in the α-glucan products from sucrose of these enzymes, i.e.
(α1→2)/(α1→4) for Gtf180-ΔN and (α1→2)/(α1→3) for GtfA-ΔN. The Gtf180-ΔN
enzyme was more efficient and produced also higher DP products than GtfA-ΔN.
Further reaction and process engineering is required to optimize conversion and
product yields.
The newly synthesized GL34 mixture maybe of interest for the food industry, more
particularly they may find application in infant foods, or in animal feed. We
therefore studied its prebiotic potential (chapter 3) by analyzing the stimulatory
effects of the GL34 mixture synthesized by Gtf180-ΔN on growth of selected gut
bacteria, including lactobacilli, bifidobacteria and commensal bacteria. The mixture
was also challenged with common carbohydrate degrading enzymes and showed
resistance to most of the tested enzymes, including α-amylase from porcine
pancreas. Bifidobacteria strains clearly grew better on the GL34 mixture than
lactobacilli and commensal bacteria. Particularly B. adolescentis grew effectively on
GL34.
When using lactose as acceptor substrate, the linkage specificity of these
glucansucrases changed to also produce (α1→2)-linkages, which is totally new for
these enzymes. Previous studies have shown that mutagenesis of residues in the
glucansucrase active site pocket may change its linkage specificity .153,154 Therefore,
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in chapter 4, we investigated the effects of mutational changes of different residues
in the acceptor substrate binding subsites on the activity and specificity of Gtf180-
ΔN when acting on lactose as acceptor substrate. The residues were selected based
on in silico docking studies of lactose into the active site pocket of the crystal
structure of Gtf180. Mutations in these residues, Q1140, W1065 and N1029,
influenced the product spectra of the GL34 mixture. Four new DP4-DP5 structures
were synthesized by mutant N1029G which favored synthesis of (α1→3) glycosidic
linkages.
Chapters 2-4 demonstrated the ability of these glucansucrases to decorate galactose-
containing compounds (lactose) and to introduce new linkage types, and indicated
that the GL34 mixture has potential as prebiotic compounds. In an attempt to
synthesize further hMOS mimics, chapter 5 studied the glucosylation of model
GOS with DP3 as acceptor substrates by Gtf180-ΔN and GtfA-ΔN. Both 4´-
galactosyl-lactose (β4´-GL) and 6´-galactosyl-lactose (β6´-GL) were used by these
enzymes and three new products were purified and structurally characterized. The
third model GOS, 3′-galactosyl-lactose (β3´-GL), was not used as an acceptor
substrate by these enzymes.
With a final aim to synthesize hMOS mimics, in chapter 6, sialylation of the GL34
mixture was carried out using trans-sialidase from Trypanosoma cruzi. Compound
F2 2-glc-lac was used as acceptor substrate by this TcTS to produce Neu5Ac-
(α2→3)-2-glc-lac with a conversion degree of 47.6 %. This enzyme also sialylated
at least five galactosylated-lactulose compounds (LGOS) structures and eleven
Vivinal GOS DP3-4 compounds. Moreover, the results revealed a strong preference
for terminal β-Gal residues to be sialylated. Only branched compounds with two
non-reducing terminal β-Gal residues were di-sialylated. Our study showed that
structures with a Gal(β1→3) terminal residue were more efficiently sialylated by
TcTS.
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Finally, in chapter 7, the results obtained in this research were summarized and
discussed. The potential use of these newly synthesized oligosaccharides for
food/feed products and their impact on future research towards hMOS mimics is
reflected.
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